Lipodepsipeptide Antibiotics: Total Synthesis and Mechanism of Action Studies Using 19F-NMR

Daptomycin is a calcium-dependent cyclic lipodepsipeptide that is used clinically in the treatment of infections caused by pathogenic Gram-positive organisms, such as methicillin-resistant Staphylococcus aureus. Another calcium-dependent cyclic lipodepsipeptide antibiotic, A54145D, also has biological activity against Gram-positive pathogenic organisms, but is not used clinically because it is approximately 4-fold less active than daptomycin. Unlike daptomycin, however, A54145D is not inhibited by lung surfactant in vitro, which makes A54145D a suitable lead compound for the development of other antibiotics that may find use in medical practice as an alternative treatment for community-acquired pneumonia caused by Streptococcus pneumoniae. While the total synthesis of daptomycin has been reported, thus allowing for an efficient method for the generation of analogs of daptomycin, no similar synthesis for A54145D was reported at the start of these studies. To facilitate the development of future antibiotics, a total synthesis of A54145D was developed employing Fmoc-based solid-phase-peptide synthesis. This synthesis enabled us to confirm that the 3-hydroxyasparagine residue has the (2S,3S)-configuration (threo), and the 3-methoxyaspartate residue has the (2S,3R)-configuration (erythro) instead of the (2S,3S)-configuration (threo). Additional analogs of A54145D and A54145A¬1 were synthesized to study the structure-activity relationships of these antibiotics, and it was determined that the threonine residue is not amenable for substitution, in that replacement of threonine caused a dramatic loss in biological activity. Furthermore, it was confirmed that the replacement of the uncommon amino acid 3-methoxyaspartate with aspartate, and 3-hydroxyasparagine with asparagine resulted in a 16-fold reduction of biological activity. To further elucidate the mechanism of action of cyclic lipodepsipeptide antibiotics, which is a matter of some controversy in the scientific literature, ten fluorinated analogs of daptomycin were constructed by semi-synthesis or total synthesis to study their interaction with model membranes using 19F-NMR. These analogs exhibited activity against Bacillus subtilis equal to or approaching that of daptomycin. 19F-NMR studies with a peptide that contained a trifluoromethyl moiety at the end of the lipid tail indicate that at concentrations of daptomycin and calcium that are representative of in vivo conditions, daptomycin does not aggregate in solution. Preliminary 19F-NMR studies with a daptomycin analog containing the CF3 group appended to the ornithine residue at position 6 in the presence of model membranes and varying amounts of calcium suggest that the ornithine residue of daptomycin interacts with biological membranes before the lipid tail does

Posterior fossa progressive multifocal leukoencephalopathy:First presentation of an unknown autoimmune disease

We present a case of a 57-year-old man who presented with progressive cerebellar dysarthria and cerebellar ataxia. Additional investigations confirmed the diagnosis of progressive multifocal leukoencephalopathy (PML) in the posterior fossa. This is a demyelinating disease of the central nervous system, caused by an opportunistic infection with John Cunningham virus. PML has previously been considered a lethal condition, but because of careful monitoring of patients with HIV and of patients using immunosuppressive drugs it is discovered in earlier stages and prognosis can be improved. Our patient had no known immune-compromising state, but further work-up revealed that the PML was most likely the first presentation of a previous untreated autoimmune disorder: sarcoidosis

Uitvoeringsagenda 2012-2016 : bron voor Groene Economie Wereldoplossingen voor werelduitdagingen

Met een duidelijke visie op de toekomst zijn vier innovatiethema's voor de komende vijf jaar op basis van economische, maatschappelijke en wetenschappelijke uitdagingen voor 2020 benoemd. Dit zijn :'Meer met minder'; ‘Voedselveiligheid en -zekerheid'; ‘Gezondheid en welbevinden’; en‘Samenwerkende waardeketen’. Deze vormen de basis voor de Uitvoeringsagenda 2012-2016. Ze zijn leidend voor alle onderdelen, namelijk de vier Innovatiecontracten (in het vervolg: Innovatieprogramma’s), het Buitenlandbeleid en de Human Capital Agenda. Met bijlage: Internationaliseringsoffensief behorende bij H4

Mechanistic studies on the effect of membrane lipid acyl chain composition on daptomycin pore formation

The final publication is available at Elsevier via https://dx.doi.org/10.1016/j.chemphyslip.2018.09.015 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/Daptomycin is a lipopeptide antibiotic that binds and permeabilizes the cell membranes of Gram-positive bacteria. Membrane permeabilization requires both calcium and phosphatidylglycerol (PG) in the target membrane, and it correlates with the formation of an oligomer that likely comprises eight subunits, which are evenly distributed between the two membrane leaflets. In both bacterial cells and model membranes, changes in the fatty acyl composition of the membrane phospholipids can prevent permeabilization. We here used liposomes to study the effect of phospholipids containing oleoyl and other fatty acyl residues on daptomycin activity, and made the following observations: (1) Oleic acid residues inhibited permeabilization when part not only of PG, but also of other phospholipids (PC or cardiolipin). (2) When included in an otherwise daptomycin-susceptible lipid mixture, even 10% of dioleoyl lipid (DOPC) can strongly inhibit permeabilization. (3) The inhibitory effect of fatty acyl residues appears to correlate more with their chain length than with unsaturation. (4) Under all conditions tested, permeabilization coincided with octamer formation, whereas tetramers were observed on membranes that were not permeabilized. Overall, our findings further support the notion that the octamer is indeed the functional transmembrane pore, and that fatty acyl residues may prevent pore formation by preventing the alignment of tetramers across the two membrane leaflets.Natural Sciences and Engineering Research Council of Canada [250265-2013,155283-2012

An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae

The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes

An amphipathic helix in Brl1 is required for nuclear pore complex biogenesis in S. cerevisiae

The nuclear pore complex (NPC) is the central portal for macromolecular exchange between the nucleus and cytoplasm. In all eukaryotes, NPCs assemble into an intact nuclear envelope (NE) during interphase, but the process of NPC biogenesis remains poorly characterized. Furthermore, little is known about how NPC assembly leads to the fusion of the outer and inner NE, and no factors have been identified that could trigger this event. Here, we characterize the transmembrane protein Brl1 as an NPC assembly factor required for NE fusion in budding yeast. Brl1 preferentially associates with NPC assembly intermediates and its depletion halts NPC biogenesis, leading to NE herniations that contain inner and outer ring nucleoporins but lack the cytoplasmic export platform. Furthermore, we identify an essential amphipathic helix in the luminal domain of Brl1 that mediates interactions with lipid bilayers. Mutations in this amphipathic helix lead to NPC assembly defects, and cryo-electron tomography analyses reveal multilayered herniations of the inner nuclear membrane with NPC-like structures at the neck, indicating a failure in NE fusion. Taken together, our results identify a role for Brl1 in NPC assembly and suggest a function of its amphipathic helix in mediating the fusion of the inner and outer nuclear membranes.publishedVersio

Incidence and trends of blastomycosis-associated hospitalizations in the United States

We used the State Inpatient Databases from the United States Agency for Healthcare Research and Quality to provide state-specific age-adjusted blastomycosis-associated hospitalization incidence throughout the entire United States. Among the 46 states studied, states within the Mississippi and Ohio River valleys had the highest age-adjusted hospitalization incidence. Specifically, Wisconsin had the highest age-adjusted hospitalization incidence (2.9 hospitalizations per 100,000 person-years). Trends were studied in the five highest hospitalization incidence states. From 2000 to 2011, blastomycosis-associated hospitalizations increased significantly in Illinois and Kentucky with an average annual increase of 4.4% and 8.4%, respectively. Trends varied significantly by state. Overall, 64% of blastomycosis-associated hospitalizations were among men and the median age at hospitalization was 53 years. This analysis provides a complete epidemiologic description of blastomycosis-associated hospitalizations throughout the endemic area in the United States